Individual nuclease activities from peripheral leukocyte subpopulations are visualized using two-dimensional gel electrophoresis with a polynucleotide in the second dimension. Proteins in cell-free extracts are separated by isoelectric focusing in a disc gel. To further resolve individual enzyme activities, a slab gel containing polynucleotide is cast on the side of the disc gel. Electrophoretic conditions for the second dimension minimize interaction between the nucleases and the substrate. No denaturing agents such as urea or SDS are necessary in either dimension. After electrophoresis, the presence of nucleases is revealed by incubation under conditions that allow substrate hydrolysis, followed by staining for polynucleotides. This procedure allows the simultaneous detection and analysis of a variety of nucleases without the necessity of multiple electrophoresis systems. Cell subpopulations from donors with chronic myelogenous leukemia exhibit differences in nuclease activities compared with those detected in equivalent cell subpopulations from normal donors. One-dimensional polynucleotide-polyacrylamide gel electrophoresis was used to examine the charge and size characteristics of the nucleases. At least some of the activities result from increased charge heterogeneity of a more limited family of nucleases.